Development of a Bioanalytical Method for Quantification of … · 2012. 5. 17. · Development of a Bioanalytical Method for Quantification of Amyloid Beta Peptides in Cerebrospinal

Development of a Bioanalytical Method for Quantification of Amyloid Beta Peptides in

Cerebrospinal Fluid

©2011 Waters Corporation 1

Joanne (乔乔乔乔安妮安妮安妮安妮) Mather

Senior Scientist

Waters Corporation

Data courtesy of Erin Chambers and Mary Lame

Background: Amyloid β Peptides

� Clinical significance of amyloid β peptides— In normal healthy individuals, Aβ peptides are

present only in small quantities as soluble monomers that circulate in cerebrospinal fluid and blood.

— Amyloid beta (Aβ) are peptides of 36–43 amino acids that are processed from the


Amyloid precursor protein

— Aβs are the main component of amyloid plaques - deposits found in the brains of patients with Alzheimer's disease

— Drug development strategies aimed at lowering production of these peptides or enhancing their clearance

o Inhibition/modulation of gamma secretase enzyme

Amyloid β Peptides for Quantification in CSF

Amyloid β 1-38

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG

MW 4132, pI 5.2, HPLC index 96

Amyloid β 1-40

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV




Amyloid β 1-42*

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA


*Aβ42 occurs more frequently and forms fibrillar aggregates far more readily than the Ab40 peptide

Internal standards are N15 labeled versions of 1-38, 1-40, and 1-42

Outline

� Introduction

� Mass Spectrometry

� Liquid Chromatography

� Choice of Standard Curve Matrix

� Solid Phase Extraction

Partial Validation


� Partial Validation

Why LCMSMS?

� Why an LCMSMS based assay?— ELISA assays not practical for

discovery, no antibodies available yet

— Challenges with ELISA assayso time consuming, expensive to

developo require separate assay for each

peptide


peptideo limited linear dynamic rangeo Possible cross reactivity

� Benefits of LC/MS/MS for peptides — LCMSMS provides single assay for

multiple amyloid peptides— Broad linear dynamic range — Accurate, precise— Universal— Faster, cheaper method

development

Specific Challenges in Developing an LCMSMS Assay for Amyloid β Peptides

� Extremely hydrophobic

� Very poor peptide solubility

� High level of aggregation —in solution & plasma

� High level of non-specific


� High level of non-specific binding (NSB)

� Low MS sensitivity—Very large and hydrophobic

—Form many low abundance fragments

� Specificity in matrix

ESI- MSMS Spectra for Amyloid β 1-42

100

Daughters of 1127ES-1.55e51126.76

1122.33

M-4H 4-

-H2O


Non-specific water loss

m/z500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450

%

0

1117.78

� Negative Ion mode ES Water loss most commonly reported MRM in literature to date

� Sensitive but not specific, Immuno-precipitation used prior to SPE

Positive Ion versus Negative Ion Detection: Specificity 1-42 in Human CSF

%

250pgmL_hCSF_032910_002x 2: MRM of 3 Channels ES- 901.2 > 897.6 (a Beta 1-42)

7.05e4

2.10

2.12

6.40

ESI-

Amyloid β 1-42


Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

1

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.500

1.122.07 5.66

5.544.274.06

2.21 3.88 5.054.894.39 5.95

6.58

7.076.62

250pgmL_hCSF_032910_002x 1: MRM of 3 Channels ES+ 1129 > 1078.5 (a Beta 1-42)

1.20e4

6.39

6.206.06

1.213.82

7.186.43

6.926.50 7.19

ESI+

Amyloid β 1-42

%

2: MRM of 3 Channels ES-825 > 821.3 (a Beta 1-38)

3.35e5

6.45

5.02

2.12

4.91

5.93

5.11

6.07

7.03

6.69

Positive Ion versus Negative Ion Detection: Specificity 1-38 in Human Plasma

ESI-

Amyloid β 1-38


Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.500

4.61 5.695.236.69

1: MRM of 3 Channels ES+ 1033.5 > 1000.3 (a beta 1-38)

2.99e4

5.93

4.96

4.83

4.222.184.60

5.10 5.81

5.41

5.94

5.99

6.676.00

6.65

7.17

6.957.28

7.947.57

7.39

ESI+

Amyloid β 1-38

Positive Ion versus Negative Ion Detection: Specificity 1-40 in Human Plasma

ESI-

Amyloid β 1-40

%

2: MRM of 3 Channels ES-864.7 > 860.9 (a Beta 1-40)

4.22e55.70

5.28

2.12

5.75

6.19


ESI+

Amyloid β 1-40

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.500

5.22

4.51 5.52

6.19

5.95

6.52

6.93

1: MRM of 3 Channels ES+ 1083 > 1053.6 (a Beta 1-40)

1.99e46.16

5.69

5.675.424.21

4.834.67 5.07

5.72

5.74

5.85

6.21

7.216.556.22

6.52 6.72 6.997.56

7.32 7.817.96

%

MRM of 2 Channels ES-1031.5 > 1026.8

3.20e4

2.48

3.36

Use of Higher m/z Precursorsand Fragments

Human Plasma Extract

4th charge state1031.5 -> 1026.8


Time0.50 1.00 1.50 2.00 2.50 3.00 3.50

%

0

0.50 1.00 1.50 2.00 2.50 3.00 3.500

MRM of 2 Channels ES-825 > 821.3

3.26e4

2.48

1.67

1.221.63

1.96 2.49

5th charge state825 -> 821.3

ESI+ MSMS Spectra for Amyloid β 1-42%

100

Daughters of 1129ES+ 1.62e41078.521053.30

1028.67

1256.501106.86

1200.08

b38

b39

b35

b40

b41

M+H 4+

b34b32


m/z500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450

%

0

1028.67

975.36

914.52

942.73

1000.65

1404.121371.29

1300.38 1437.73

b33

b34

b35

b36

3+ fragments4+ fragments

b39 b40b35 b38

� ESI+ Sequence fragments - more specific

� High masses - more specific

Amyloid β Peptide 1-42:Identification of Fragments with BioLynx In Silico Fragmentation Model

Fragment choice is 4+ b 40 ion


Xevo™ TQ-S MSMS Conditions

Xevo™ TQ-S MS operated in ESI+ modeMRM Transitions:

Peptide Name

Precursor

Ion 4+

Product

Ion 4+

Product

Ion ID

Cone

voltage (V)

Collision

energy (eV)

Amyloid β 1-38 1033.5 1000.3 b 36 33 23

Amyloid β 1-38 N15 IS 1046 1012.5 30 22

Amyloid β 1-40 1083 1053.6 b 39 33 25

Amyloid β 1-40 N15 IS 1096 1066.5 35 22


Amyloid β 1-40 N15 IS 1096 1066.5 35 22

Amyloid β 1-42 1129 1078.5 b 40 28 30

Amyloid β 1-42 N15 IS 1142.5 1091.5 35 28

Outline

� Introduction





Partial Validation



Column: 2.1 X 150mm, ACQUITY BEH C18 300Å, 1.7µmMobile phase A: 0.3% NH4OH by volume, or 0.1% absoluteMobile Phase B: 90/10 ACN/mobile phase ATemperature: 50° CSNW: 65/25/10 ACN/water/NH4OHSNW volume: 600 µLWNW: 90/10 water/ACN + 0.3% NH4OHWNW volume: 600 µLFlow rate: 0.2 mL/minInjection mode: partial loop

UPLC® Conditions: Final Method

Gradient Table


Injection mode: partial loopInjection Volume: 10 µLInjection Solvent for standards: SPE elution solvent diluted with water;SPE elution solvent = 75% ACN, 15% water, 10% NH4OH

in 50/50 elution solvent/water

100

3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

100

12pt5pgmL_abetas_072610_001 Sm (SG, 3x5) MRM of 3 Channels ES+ 1129.5 > 1078.9 (Amyloid Beta 1-42)

5.58e3Area

5.81125.0

12pt5pgmL_abetas_072610_001 Sm (SG, 3x5) MRM of 3 Channels ES+ 1083.3 > 1054 (Amyloid Beta 1-40)

5.58e3Area

Xevo TQ-SLOD: 12.5 pg/mL solvent standard

Amyloid β 1-42Peak Area 125


Time3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

100

3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

Area

5.6298.0

12pt5pgmL_abetas_072610_001 Sm (SG, 3x5) MRM of 3 Channels ES+ 1033.75 > 1000.75 (Amyloid Beta 1-38)

5.58e3Area

5.39;129.9

10µL injection



Representative Chromatogram:Extracted Spiked Artificial CSF

Amyloid β 1-38

Amyloid β 1-40

Amyloid β 1-42

4.60e4

5.85

5.60

6.03

Peak Widths 2-3 seconds


Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

2

5.304.89

6.87

6.31

7.65

7.97

Representative Chromatography: Extracted Basal (Endogenous) Level Human CSF Sample

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

1

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

hCSF_pool1_basal_040210_001 MRM of 6 Channels ES+ 1142.5 > 1091.5 (a Beta 1-42 N15)

2.77e46.02

6.01 6.06

6.766.53 7.47 7.69

hCSF_pool1_basal_040210_001 MRM of 6 Channels ES+ 1129 > 1078.5 (a Beta 1-42)

8.54e3

6.03

6.01

1.851.25 5.885.424.90

6.05

7.186.616.47

6.187.07 7.817.67 7.91

hCSF_pool1_basal_040210_001 MRM of 6 Channels ES+ 1096 > 1066.5 (a Beta 1-40 N15)5.85

1-42 0.49ng/mL

1-42 N15 IS

1-40 N15 IS


Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

1096 > 1066.5 (a Beta 1-40 N15)6.29e4

5.85

4.80

5.87

5.896.37 6.58


1.16e5

5.85

5.52

5.88

hCSF_pool1_basal_040210_001 MRM of 6 Channels ES+ 1046 > 1012.5 (a Beta 1-38 N15)

4.05e4

5.61

5.36

5.62

5.677.076.676.46

hCSF_pool1_basal_040210_001 MRM of 6 Channels ES+ 1033.5 > 1000.3 (a beta 1-38)

2.31e45.61

5.271.19

5.63

6.756.395.67 5.90

7.987.02

1-38 1.63ng/mL

1-40 3.24 ng/mL

1-38 N15 IS

1-40 N15 IS

Outline

� Introduction





Partial Validation



Choice of Standard Curve Matrix

� Artificial CSF chosen as surrogate matrix

� Lower cost

� Higher availability

� Does not influence Aβ quantification

� No spinal tap required

No standard addition calculations


� No standard addition calculations

� Artificial CSF

� Issues with recovery caused by non-specific binding

� 5% Rat plasma added

� Plasma increased recovery & reproducibilty

� Plasma also helps to mimic real CSF (similar proteins)

Average Baseline Levels in Pooled Human CSF: Comparison of Results with Different Standard Curve Preparations

Amyloid β 1-38

Conc.(ng/mL)

Source 1

Conc. (ng/mL)

Source 2

Conc from curve in

art CSF 0.82 0.826

Conc from curve in

human CSF 0.679 n/a

Amyloid β 1-40

Conc.(ng/mL)

Source 1

Conc. (ng/mL)

Source 2

Conc from curve in

art CSF 3.713 3.136

Conc from curve in

human CSF 4.335 n/a

� Excellent correlation between artificial CSF & human CSF

� Standard curves prepared from either artificial CSF or by over-spiking human CSF


average 0.750

std deviation 0.100

RSD 13.302

average 4.024

std deviation 0.440

RSD 10.930

Amyloid β 1-42

Conc.(ng/mL)

Source 1

Conc. (ng/mL)

Source 2

Conc from curve in

art CSF 1.105 0.763

Conc from curve in

human CSF 1.189 0.804

average 1.147 0.784

std deviation 0.059 0.029

RSD 5.178 3.700

*Human CSF corrected for basal level

Outline

� Introduction





Partial Validation



Sample Extraction: Pretreatment

Sample Pretreatment

Spike human or artificial CSF + 5% rat plasma samples, mix

Equilibrate samples at room temperature for 30 minutes

Remove 50 µL spiked artificial CSF (to which 5% rat plasma was added) for standard curves, or human/monkey CSF (basal level or over-spike


for standard curves, or human/monkey CSF (basal level or over-spike QC’s)

Add 50µL 5M guanidine HCl (denatures & eliminates protein binding)

Shake samples at room temperature for 45 minutes

Add 50µL 4% H3PO4 in water, mix

Oasis® MCX µElution Plate

Condition: 200 µL MeOH

Equilibrate: 200 µL 4% H3PO4

Load: 150 µL diluted sample (pretreated sample: 50 µL human CSF, 50µL 5M guanidine HCl, 50µL 4% H3PO4 in water)

Sample Extraction: Final Method

Amyloid β

Peptide

% SPE

Recovery

1-38 94%

1-40 92%

1-42 90%


5M guanidine HCl, 50µL 4% H3PO4 in water)

Wash 1: 200 µL 4% H3PO4

Wash 2: 200 µL 10% ACN in water (by volume)

Elute: 2 X 25 µL 75/10/15 ACN/conc. NH4OH/water (by volume)

Dilute: 25 µL water

Inject: 10 µL

Key Attributes for High Recovery Extraction

� Wash with no more than 10% ACN— Removes polar interferences— Does not impact recovery of 1-38 (earliest eluting/least

hydrophobic of these 3 amyloid β peptides)

� Elute with no less than 75% ACN— Provides required elutropic strength for 1-40 and 1-42 (the

more hydrophobic of these 3 peptides)


more hydrophobic of these 3 peptides)

� Elute with no less than 10% NH4OH— Provides required solubility to fully elute 1-42 (least

soluble/most hydrophobic of these 3 peptides)

� Addition of rat plasma to artificial CSF to eliminate non-specific binding increases SPE recovery in this matrix from 60% on average to >90%

� Guanidine HCl denaturation— Improves reproducibility of method; eliminates aggregation

and protein binding

Outline

� Introduction





Partial Validation



Summary of Samples Extracted

� Standard curves prepared in artificial CSF + 5% Rat plasma— Calibration points are 0.025, 0.05, 0.1, 0.25, 0.35, 0.5, 0.75,

1, 5, 7.5, and 10 ng/mL

� Basal levels in human CSF— 3 individual sources pooled human CSF samples, 1 source

cynomalgous monkey


cynomalgous monkey

— 6 replicates from each source

� QC samples— Prepared in each of the human CSF matrices

— 7 concentrations: 0.04, 0.075, 0.15, 0.2, 0.8, 2 and 6 ng/mL over-spike

— N=3 for each concentration, from each source of CSF

Representative Chromatogram: Basal Levels of Amyloid β 1-38 in Human and Monkey CSF

%

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0


2.31e4

5.61

5.271.19

5.63

5.64

6.756.395.795.90 7.987.02


2.96e4

5.61

5.60

Human CSF, pooled sample 1



Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

1

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.500

1.12 5.262.12

5.66

6.746.546.235.91 7.13


1.33e4

5.61

5.60

3.992.121.22

5.65

6.746.505.94

6.21 7.866.96 7.62

cyno_CSF_pool_basal_040210_001 MRM of 6 Channels ES+ 1033.5 > 1000.3 (a beta 1-38)

2.43e4

5.62

5.59

5.042.121.101.295.23 7.826.966.36

6.185.74 6.79 7.71


Monkey CSF, pooled sample 1


0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0


1.16e5

5.85

5.52

5.88


1.47e5

5.85




Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0


8.91e4

5.86

cyno_CSF_pool_basal_040210_001 MRM of 6 Channels ES+ 1083 > 1053.6 (a Beta 1-40)

1.37e5

5.86

6.57




%

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0


6.61e3

6.03

5.931.85

1.255.42

5.71 7.186.59

6.477.817.71 7.91


8.77e3

6.04




Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

-1

99

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

2

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.501

5.911.85 5.743.91 4.98

6.416.30

6.64 7.886.87


4.87e3

6.04

5.901.101.22

1.89 5.16 6.426.10 6.74 7.486.927.93

cyno_CSF_pool_basal_040210_001 MRM of 6 Channels ES+ 1129 > 1078.5 (a Beta 1-42)

8.13e3

1.86

1.16

6.04

2.09 4.76 4.91 5.94 6.366.47 7.866.88 7.507.24



Baseline Levels of Amyloid β in Pooled Human and Monkey CSFAmyloid Beta 1-38

Replicate #

Human CSF

Pool 1

ng/mL

Human CSF

Pool 2 ng/mL

Human CSF

Pool 3

ng/mL

Cyno CSF

Pool 1 ng/mL

1 1.585 2.354 1.014 1.713

2 1.650 2.103 1.371 1.605

3 1.614 2.464 0.950 1.947

4 1.657 1.939 1.608 1.541

5 1.820 2.158 1.471 1.675

6 1.486 1.995 1.167 1.644

Mean 1.635 2.169 1.264 1.688

Std. Deviation 0.110 0.204 0.262 0.140

Amyloid Beta 1-42

Replicate #

Human CSF

Pool 1

ng/mL

Human CSF

Pool 2 ng/mL

Human CSF

Pool 3

ng/mL

Cyno CSF

Pool 1 ng/mL

1 0.519 0.616 0.421 0.675

2 0.421 0.656 0.481 0.621

3 0.542 0.644 0.534 0.623

4 0.471 0.567 0.348 0.659

5 0.476 0.573 0.487 0.700

6 0.561 0.713 0.510 0.688

Mean 0.498 0.628 0.463 0.661

Std. Deviation 0.052 0.055 0.068 0.033


% CV 6.7 9.4 20.7 8.3

Amyloid Beta 1-40

Replicate #

Human CSF

Pool 1

ng/mL

Human CSF

Pool 2 ng/mL

Human CSF

Pool 3

ng/mL

Cyno CSF

Pool 1 ng/mL

1 3.083 4.031 2.541 3.699

2 3.391 3.776 2.593 3.989

3 3.292 3.598 2.580 3.525

4 2.884 3.533 2.612 3.956

5 3.131 3.230 2.508 3.284

6 3.656 3.619 2.490 3.595

Mean 3.240 3.631 2.554 3.675

Std. Deviation 0.269 0.266 0.049 0.268

% CV 8.3 7.3 1.9 7.3

% CV 10.4 8.7 14.7 5.1

Representative Standard Curve: Amyloid β 1-42

Compound name: Amyloid Beta 1-42

Correlation coefficient: r = 0.998799, r^2 = 0.997600

Calibration curve: 0.887923 * x + 0.0130161

Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Residual

0.0

10.0


Conc0.0 2.0 4.0 6.0 8.0 10.0

Response

0.00

5.00

ConcResidual

-10.0

0.0

Representative Standard Curve: Amyloid β 1-42

Name Type Std. Conc RT Area IS Area Response Conc. %Dev

blank artificial CSF 5.73 19.7 7.0

50 pg/mL artificial CSF Standard 0.05 5.71 230.4 3620.5 0.064 0.057 14

100 pg/mL artificial CSF Standard 0.1 5.71 390.8 3585.1 0.109 0.108 8.1

250 pg/mL artificial CSF Standard 0.25 5.71 778.3 3737.3 0.208 0.220 -12

350 pg/mL artificial CSF Standard 0.35 5.71 1267.3 3693.8 0.343 0.372 6.2

500 pg/mL artificial CSF Standard 0.5 5.71 1494.7 3566.8 0.419 0.457 -8.5




1 ng/mL artificial CSF Standard 1 5.71 3166.8 3792.5 0.835 0.926 -7.4

5 ng/mL artificial CSF Standard 5 5.72 14773.9 3148.3 4.693 5.270 5.4

7.5 ng/mL artificial CSF Standard 7.5 5.72 24576.9 3877.0 6.339 7.125 -5

10 ng/mL artificial CSF Standard 10 5.72 33343.3 3662.5 9.104 10.238 2.4

Representative Basal Levels and QC’s in Human CSF: Amyloid β 1-42

Name Type Std. Conc RT Area IS Area Response Conc. %Dev

Human CSF 1 Basal 5.72 1932.0 4858.4 0.398 0.433

Human CSF 1 Basal 5.72 2110.6 4933.2 0.428 0.467

Human CSF 2 Basal 5.72 1151.3 5522.8 0.208 0.220

Human CSF 2 Basal 5.72 1145.6 5636.5 0.203 0.214

human CSF 1 QC 40 pg/mL QC 0.498 5.72 2341.5 5389.8 0.434 0.475 -4.7

human CSF 2 QC 40 pg/mL QC 0.266 5.72 1359.5 5107.3 0.266 0.285 7.2


human CSF 2 QC 75 pg/mL QC 0.226 5.72 1453.1 4944.0 0.294 0.316 40









human CSF 1 QC 2 ng/mL QC 2.458 5.73 11006.4 5201.9 2.116 2.368 -3.7

human CSF 2 QC 2 ng/mL QC 2.226 5.73 10249.0 5025.8 2.039 2.282 2.5

human CSF 1 QC 6 ng/mL QC 6.458 5.73 28977.8 5029.9 5.761 6.474 0.2

human CSF 2 QC 6 ng/mL QC 6.226 5.73 26727.1 4848.4 5.513 6.194 -0.5

Average Deviation Values for all Overspike QC Samples

QC 0.04

ng/mL

QC 0.075

ng/mL

QC 0.15

ng/mL

QC 0.2

ng/mL

QC

0.8ng/mL

QC 2

ng/mL

QC 6

ng/mL

Amyloid β 1-38 Human

CSF 1 and 2 2.3 5.8 -3.2 7.3 14.8 5.1 13.1




CSF 1 and 2 -0.8 -3.2 -1.9 2.5 -2.6 -4.2 -3.8


CSF 1 and 2 1.3 13.4 -3.6 5.6 2.0 -0.6 -0.2

Conclusions

� Single flexible LC/MS/MS platform developed for simultaneous quantification of multiple amyloid peptides

� Highly selective sample preparation based on mixed-mode SPE

� Improved MS selectivity using positive ion mode and sequence ion fragments

� High sensitivity using new MS platform


� High sensitivity using new MS platform

� Highly reproducible, accurate, and precise

� Sample pretreatment and choice of SPE and LC solutions eliminate handling (NSB, losses, etc.) problems

Quantitation of amyloid beta peptides Aβ(1-38), Aβ(1-40), and Aβ(1-42)

in human cerebrospinal fluid by ultra-performance liquid

chromatography-tandem mass spectrometry.

Authors: Mary E Lame, Erin E Chambers, Matthew Blatnik

Analytical biochemistry. 08/2011; 419(2):133-9.

Development of a Bioanalytical Method for Quantification of … · 2012. 5. 17. · Development of a Bioanalytical Method for Quantification of Amyloid Beta Peptides in Cerebrospinal

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